Peptide antagonists of CGRP-receptor superfamily and methods of use

ABSTRACT

This invention relates to antagonists of calcitonin gene related peptide and in particular the invention relates to amino terminal modifications to peptides to improve their ability to bind to a member of the CGRP-receptor superfamily.

[0001] This invention supported in part by NIH Grant No. HL51131. Thegovernment may have certain rights to this invention.

FIELD OF THE INVENTION

[0002] This invention relates to the field of vasoactive compounds andtheir antagonists. In particular, this invention relates to antagonistsof the vasoactive peptide CGRP and other members of the CGRPsuperfamily.

BACKGROUND OF THE INVENTION

[0003] The calcitonin gene related peptide (CGRP) is a sensoryneuropeptide with potent vasodilatory and cardiotonic action asdescribed in U.S. Pat. No. 4,530,838 to Evans et al. The peptide existsin two forms (denoted α and β). α-CGRP is produced by the calcitoningene (Amara et al. Nature 298:240-244, 1982 and Rosenfeld et al. Nature304:129-135, 1983) while β-CGRP is the product of a separate gene (Amaraet al. Nature 298:240-244, 1985 and Steenbergh et al. FEBS Lett.183:403-407, 1985). The human β-form and α-form differ by three aminoacids.

[0004] CGRP is concentrated in those areas of the body receiving sensoryinput from the dorsal horn with limited amounts associated withautonomic input. The peptide is present in the brain in the nuclei ofsensory and motor cranial nerves and in cell bodies in the hypothalamus,preoptic area, ventromedial thalamus, hippocampus, and the like. CGRP isfound in both sensory and motor nerves of the peripheral nervous system.The peptide is found in the skin, blood vessels, heart, gastrointestinaltract, tongue, esophagus, pancreas, salivary glands, lungs, kidney andother organs (Poyner, D. Pharmac. Ther. 56:23-51, 1992).

[0005] The release of CGRP from sensory nerve endings in inflammatoryreactions can result in the local acceleration of microhemodynamicchanges including vasodilation and permeability of the microcirculationresulting in plasma exudation and the release of humoral factors andinflammatory cells to the site of injury. CGRP has been used as avasodilator in animal models of subarachnoid hemorrhage and in trialsinvolving human subjects with congestive heart failure. CGRPadministration produced hypotension associated with moderate tachycardiain hypertensive humans (Jian et al. Chin. Med. J 102:897-901, 1989).CGRP has also been used as a potent dilator of the coronary circulation(Ezra et al., Eur. J Pharmacol., 1987). In contrast to nitrates, whichhave also been used as vasodilators, CGRP results in dilation by bothendothelium-dependent and endothelium-independent mechanisms. Also, incontrast to nitrates, such as sodium nitroprusside, tolerance to CGRPhas not been noted (Foulkes et al. Regul. Pept. 25:25-36, 1989). CGRPhas been demonstrated to improve the ability of patients to participatein exercise programs in patients with chronic stable angina (Uren et al.Cardiovasc. Res. 27:1477-1481, 1993).

[0006] CGRP has a number of problems as a therapeutic. CGRP isnonselective, inactive in oral form, generally has a short duration ofaction and has a number of side effects that can include uncontrolledhypotension (Feuerstein et al. Can. J. Physiol. Pharmacol. 73:1070-1074,1995).

[0007] CGRP has been implicated in migraines, diabetes, sepsis andinflammation. Migraines are noted for the strength of the headache thatensues with its pathology. Most believe that the headache associatedwith migraines results from the profound cerebral vasodilation. CGRPcontaining nerve fibers innervate cerebral and dural vessels where CGRPis believed to prolong vasodilation. (Moskowitz Trends Pharmacol. Sci.13:307-311, 1992). Elevated CGRP was found in the jugular vein blood ofpatients with migraines during a period where the patients complained ofmigraine symptoms, including headaches. For these reasons, CGRPantagonists have been proposed as a method for blocking cerebrovascularCGRP receptors and thus blocking the vasodilation causing migraine.

[0008] CGRP has also been postulated to be a potent indirect antagonistof insulin effects on glucose metabolism and CGRP was shown to produceinsulin resistance in rat studies (Molina et al. Diabetes 39:260-265,1990). For this reason CGRP has recently been implicated in Type IIdiabetes mellitus and to abnormalities associated with carbohydratemetabolism and hyperglycemia. CGRP has also been implicated in thehemodynamic derangements associated with endotoxemia and sepsisresulting from a variety of infectious diseases. Animals exposed tolipopolysaccharide (LPS) had elevated levels of CGRP and this coincidedwith hypotension and tachycardia in these animals (Joyce et al. Surgery108:1097-1101, 1990 and Griffin et al. Circ. Shock 38:50-54, 1992).

[0009] CGRP binds to a number of different receptors, some of which havebeen characterized. Radioligand binding studies to assess CGRP affinityfor CGRP receptors is well known in the literature (Poyner, D. R.Pharmac. Ther. 56:23-51, 1992). As stated in Poyner et al., a problemassociated with studies to identify CGRP receptors is that lack ofsuitable CGRP receptor binding analogs and it is accepted that the useof CGRP antagonists is a useful way of classifying CGRP receptors. Theart recognizes that there are a limited number of antagonists and thatit would be desirable to have more CGRP antagonists to further classifyand understand CGRP activity.

[0010] Molecules that compete for the CGRP receptor are known. Theseinclude, for example, [Tyr°]CGRP(28-37) and CGRP(8-37). Other moleculesthat compete for the CGRP receptor include peptides comprising thesequence of CGRP but that lack at least the first five amino acids ofthe CGRP amino acid sequence. [Tyr°]CGRP(28-37) was able to antagonizeall forms of CGRP tested but with different potencies. Other moleculesthat compete for the CGRP receptor are provided elsewhere in thisdisclosure.

[0011] CGRP antagonists includes peptides from CGRP including aminoacids 8-37 of β- CGRP (Chiba et al. Am. J. Physiol. 1989) having theamino acid sequence: THRLAGLLSRSGGMVKSNFVPTNVGSKAF (SEQ ID NO: 1) andpeptides from α-CGRP including amino acids 8-37 and having the aminoacid sequence THRLAGLLSRSGGMVKSNFVVPTNVGSKAF. β-CGRP(8-37) (SEQ ID NO:2)has been used to counteract the effects of CGRP. For example, CGRP(8-37)has been shown to reverse the hypotension and tachycardia produced byadministration of LPS to rats (Huttemeir, et al. Am. J. Physiol.265:H767-H769, 1993). In addition, CGRP(8-37) has some activity againstamylin (Gardiner et al. Diabetes 40:948-951, 1991). The affinity forCGRP(8-37) varies between tissues. For example, data indicates that theaffinity of CGRP(8-37) for mesenteric artery, kidney, heart and skeletalmuscle is somewhat higher than the affinity of CGRP(8-37) for adipocytesand descending colon (Poyner, D. Trends in Pharm. Sci. 16:424-428,1995).

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1A is a graph demonstrating the inhibition of α-CGRP bindingto coronary artery membranes in the presence of exemplary peptides,according to this invention. The symbol ▪ indicates h-αCGRP (8-37) ΔN-benzyl-h-α-CGRP (8-37) ⋄ N-benzoyl-h-α-CGRP (8-37) ∘ dibenzyl-h-α-CGRP(8-37). FIG. 1B is a graph showing the inhibition of CGRP binding by theβCGRP (8-37) derivatives to coronary artery membranes in the presence ofexemplary peptides according to this invention. The symbol ▪ indicatesh-βCGRP (8-37) Δ N-benzyl-h-βCGRP (8-37) ⋄ N-benzoyl-h-βCGRP (8-37) ∘dibenzyl-h-βCGRP (8-37).

[0013]FIG. 2 illustrates the antagonist effect of αCGRP(8-37) modifiedpeptides on h-αCGRP-induced relaxation of capsaicin-treated pig coronaryartery.

[0014]FIG. 2A illustrates the antagonistic effect of h-αCGRP(8-37). Thesymbol □ indicates Control+h-αCGRP (8-37) ▪ 3×10⁻⁶ M ▴ 1×10⁻⁵ M  3×10⁻⁵M.

[0015]FIG. 2B illustrates the antagonistic effect ofN-benzoyl-h-αCGRP(8-37). The symbol ▪ indicatesControl+N-benzoyl-h-αCGRP (8-37) □ 1×10⁻⁷ M Δ 3×10⁻⁷ M ⋄ 1×10⁻⁶ M ∘3×10⁻⁶ M.

[0016]FIG. 2C illustrates the antagonistic effect ofN-benzyl-h-αCGRP(8-37). The symbol ▪ indicates Control+N-benzyl-h-αCGRP(8-37) Δ 1×10⁻⁶ M ⋄ 3×10⁻⁶ M ∘ 1×10⁻⁵ M.

[0017]FIG. 2D illustrates the antagonistic effect ofdibenzyl-h-αCGRP(8-37). The symbol ▪ indicates Control+dibenzyl-h-αCGRP(8-37) Δ 3×10⁻⁷ M ⋄ 1×10⁻⁶ M ∘ 3×10⁻⁶ M.

SUMMARY OF THE INVENTION

[0018] The present invention relates to vasoactive peptides having thegeneral formula:

R¹—X—Z

[0019] wherein Z is a vasoactive peptide, R1 is an organic group, X is

[0020] and wherein R2 and R3 are independently H or an organic group andn is a whole integer between 1 and 10.

[0021] In a preferred embodiment, Z is a peptide fragment of CGRP. Inone embodiment the amino acid sequence of CGRP is SEQ ID NO:3 and inanother the amino acid sequence of CGRP is SEQ ID NO:4. Preferably theCGRP is human CGRP and preferably either α-CGRP or β-CGRP. In apreferred embodiment the peptide fragment is CGRP(8-37), Tyr°CGRP(28-37), or CGRP(12-37). In another embodiment the peptide fragmentis a CGRP antagonist and preferably selected from the group of peptidesor peptide fragments from amylin or adrenomedullin that bind to one ormore CGRP receptors. In another embodiment, the peptide fragments arepeptide fragments with CGRP antagonist activity selected from CGRPreceptor binding peptides preferably having at least 15 amino acids fromthe amino acid sequences of SEQ ID NOS:5-19.

[0022] Also preferably, R¹ is an aromatic group, a heterocyclic group oran alkyl group and R² and R³ are independently H, an aromatic group oran alkyl group. In one embodiment, R¹ is a C1-C18 aromatic group, or aC1-C4 alkyl group and in another embodiment, R¹ is a fluoroalkyl group.In another embodiment, R¹ is a C5-C10 aromatic group, a C5-C9heterocyclic group or a C1-C18 alkyl group.

[0023] Preferably R² and R³ are independently H, a C1-C4 alkyl group ora phenyl moiety. Preferably, R² and R³ are independently H or a C5-C10aromatic group or a C1-C4 alkyl group.

[0024] In one embodiment, R¹ has the general formula:

[0025] and R⁴-R⁸ are each independently selected from the group of H,fluoro, chloro, bromo, iodo, nitro, nitrile (cyano), amino, N-methylamino, N,N-dimethyl amino, hydroxy, methoxy, thiomethoxy (S-methyl),methyl, ethyl, n-propyl, iso-propyl, -butyl, iso-butyl, sec-butyl,tert-butyl, trifluoromethyl, trifluoromethoxy, vinyl, acetamido, phenyl,toluyl, and methoxyphenyl. In a preferred embodiment, R⁶ istrifluoromethyl and one or more of R⁴, R⁵, R⁷ and R⁸ is F.

[0026] In another embodiment of the invention, R¹ is

[0027] and Y is selected from the group consisting of O, NH, and S.

[0028] In yet another embodiment, R¹ is selected from the groupconsisting of:

[0029] and X is C or N.

[0030] In another aspect of this invention, the peptides of thisinvention are selected from the group consisting of N-α-benzoyl-α-CGRPreceptor antagonist peptides, N-α-benzyl-β-CGRP receptor antagonistpeptides, N-α-benzoyl-β-CGRP receptor antagonist peptides, N-α-benzyl-αCGRP receptor antagonist peptides, N-α-benzyl-[(4′benzyl-His¹⁰]-α-CGRPreceptor antagonist peptides, [(4′benzyl-His¹⁰]-β-CGRP receptorantagonist peptides, N,N-dibenzyl-α-CGRP and N,N-dibenzyl-β-CGRP.

[0031] The invention also relates to a method for inhibiting CGRPbinding to one or more CGRP receptors comprising the step of contactingan effect amount of a peptide of this invention with the CGRP receptorwherein R¹ of the peptide is an aromatic group, a heterocyclic group oran alkyl group and R² and R³ of the peptide are independently H, anaromatic group or an alkyl group. The receptor can be cell free or cellassociated. The receptor can be in a cell in culture or in a cell aspart of a tissue of an animal, including humans.

[0032] The invention further relates to an assay for identifying CGRPantagonists comprising the step of: combining at least one peptide ofthis invention and a candidate CGRP antagonist with a CGRP receptor andcomparing binding of the peptide to the CGRP receptor and binding of thecandidate antagonist to the CGRP receptor wherein binding of thecandidate antagonist to the CGRP receptor in the presence of the peptideidentifies a CGRP antagonist.

[0033] The invention also relates to a method for inhibiting CGRPactivity comprising the step of administering an effective amount of apeptide of this invention to a cell wherein the cell comprises a CGRPreceptor.

[0034] In yet another aspect of this invention, the invention relates toa method for identifying a CGRP receptor in a cell sample comprising thestep of detecting binding of a peptide of this invention to a cell andisolating and/or characterizing the receptor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention relates to modified peptides that serve asvasoactive peptide antagonists for the CGRP receptor. In a preferredembodiment, this invention relates to peptides with amino terminalmodifications wherein the peptide functions as a CGRP antagonist.

[0036] In one embodiment, this invention relates to vasoactive peptideswith the following general formula:

R¹—X—Z

[0037] wherein Z is a vasoactive peptide fragment, R¹ is an organicgroup, X is

[0038] and wherein R² and R³ are independently H or an organic group andn is a whole integer between 1 and 10.

[0039] The term “vasoactive peptide” refers to peptides that are capableof causing vasoconstriction or vasodilatation of blood vessels and apeptide capable of binding to a CGRP receptor refers to peptides,preferably of at least 15 amino acids in length that have CGRP receptorbinding activity. For purposes of this invention, a CGRP receptor is anisolated or cell associated receptor with CGRP binding activity.

[0040] A variety of vasoactive peptides or peptide fragments thatfunction as CGRP antagonists are known in the art and these include, butare not limited to, CGRP receptor- binding peptide fragments of CGRP,including the α and β forms of CGRP and peptides of adrenomedullin andamylin. CGRP has been isolated from a variety of animals including, butnot limited to humans (β-CGRP, SEQ ID NO:3; α-CGRP; SEQ ID NO:4, Poyner,D. R. Pharmac. Ther. 56:23-51, 1992), rats (β-CGRP, SEQ ID NO:5; α-CGRP.SEQ ID NO:6, Poyner, supra), chickens (SEQ ID NO:7, Poyner, supra),rabbits (SEQ ID NO:8, Eysselein et al. Peptides 12:289-295, 1991), pigs(SEQ ID NO:9, Kimura, S. et al. Neuropeptides 9:75-82,1987), sheep (SEQID NO:10, Miyata et al. Biochem. Biophys. Res. Commun. 187:1474-1479,1992), cows (SEQ ID NO:1 1 Collyear, K. et al. J. Mol. Endocrinol.6:147-152, 1991), salmon (SEQ ID NO:12, Jansz, et al. Ann. N. Y Acad.Sci. 657:63-69, 1992) and frogs (SEQ ID NO:13 Esneu et al. Endocrinol.135:432-430,1994). Adrenomedullin has been isolated from a variety ofsources including human (SEQ ID NO:14 Kitamura, K. et al. Biochem.Biophys. Res. Commun. 195:921-927, 1993) and rat (SEQ ID NO:15 Sakata,J. et al. Biochem. Biophys. Res. Commun. 195:921-927, 1993). Amylin hasalso been isolated from a variety of sources including, but not limitedto, human (SEQ ID NO: 16 Westernark, P. et al. Proc. Natl. Acad. Sci.USA 84:3881-3885, 1987) and rat (SEQ ID NO:17 Leffert, J. D. et al.Proc. Natl. Acad. Sci. USA 86:3127-3130, 1989).

[0041] Antagonists of the CGRP receptor include a variety of peptidesincluding peptide fragments from CGRP peptides including, but notlimited to, CGRP (8-37), CGRP (28-37) including Tyr° CGRP (28-37), andCGRP (12-37). Other CGRP antagonists include h-α-CGRP (9-37), h-α-CGRP(10-37), h-α-CGRP (11-37) (Mimeault, M. et al., J. Med Chem.35:2163-2168,1992). Still other CGRP antagonists include [Ala⁹]-h-α-CGRP(8-37), [Ala¹⁰]-h-α-CGRP (8-37), [Ala¹¹]-h-α-CGRP (8-37), and[Ala¹²]-h-α-CGRP (8-37). Additional CGRP antagonists includeh-α-CGRP(19-37), h-α-CGRP(23-37) and acetyl-h-α-CGRP(19-37) (Rovero, P.et al. Peptides 13:1025-1027, 1992).

[0042] Amylin antagonist peptides are known and a number of these withCGRP receptor binding activity are provided in U.S. Pat. No. 5,625,032to Gaeta et al., and U.S. Pat. No. 5, 580,953 to Albrecht et al.Preferred amylin antagonist peptides include:

[0043] human amylin (8-37)

[0044] HATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH₂ (SEQ ID NO: 18),

[0045] rat-amylin(8-37)

[0046] H-ATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH₂) SEQ ID NO: 19),

[0047] and Acetyl-Rat-Amylin (8-37) (Deems et al. Biochem. Biophys. Res.Commun. 181:116-120, 1991).

[0048] Still other amnylin antagonists that can be tested for CGRPreceptor binding activity include:

[0049] AC187 (SEQ ID NO:20)

[0050] (Acetyl-VLGKLSQELHKLQTYPRTNTGSNTY-NH₂, Beaumont et al. Br. J.Pharmacol. 115:713-715, 1995),

[0051] AC253 (SEQ ID NO:21)

[0052] (Acetyl-LGRLSQELHRLQTYPRTNTGSNTY-NH2), and

[0053] AC625 (SEQ ID NO:22)

[0054] (Acetyl-ATQRLANELVRLQTYPRTNVGSNTY-NH2 both Prickett, K. S. et al.in Peptides Chemistry and Biology, eds. Kaumaya, P. T. P and Hodges, S.Mayflower Scientific Ltd., Kingswinford, UK. 1996).

[0055] Preferred adrenomedullin-derived antagonists include:

[0056] h-adrenomedullin (22-52) (SEQ ID NO:23)

[0057] (TVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-NH₂ Watanabe, et al. Endocrinol.135:2454-2458, 1994 and Champion et al. Am. J PhysioL 272:R234-242,1997),

[0058] In addition, the modifications of this invention can beincorporated into other polypeptides with vasoactivity and having CGRPreceptor binding activity, such as relaxin, a molecule structurallyrelated to amylin (Cooper et al., Proc. Natl. Acad. Sci. USA85:7763-7766, 1988). Moreover, substituted peptides of amylin, CGRP orother vasoactive peptides are described in U.S. application Ser. No.275,475.

[0059] In general, the vasoactive peptides of this invention include acarboxyamide at the C-terminus. Alternatively, the peptides of thisinvention can include a free carboxyl group at the terminus.Abbreviations for peptide termini are as follows: “H—” refers to a freeamino group, “—OH” refers to a free carboxyl group and “—NH₂” refers toa carboxyamide. The term “vasoactive peptide” as used herein refers topeptides with physiological activity, particularly, but not necessarilysolely, directed in activity to the vasculature system and preferablypeptides with CGRP antagonist activity. In general, the peptides of thisinvention exhibit greater activity when a carboxyamide is positioned atthe terminus of the peptide. Methods for preparing peptides withC-terminal amide groups are known in the art and, in one example,described in U.S. Pat. No. 5,503,989 to Bibbs et al.

[0060] The amino acid designations used throughout this patentapplication include the standard amino acid designations: A or Ala forAlanine, C or Cys for Cysteine, D or Asp for Aspartic acid, E or Glu forGlutamic acid, F or Phe for Phenylalanine, G or Gly for Glycine, H orHis for Histidine, I or Ile for Isoleucine, K or Lys for Lysine, L orLeu for Leucine, M or Met for Methionine, N or Asn for Asparagine, P orPro for Proline, Q or Gln for Glutamine, R or Arg for Arginine, S or Serfor Serine, T or Thr for Threonine, V or Val for Valine, W or Trp forTryptophan and Y or Tyr for Tyrosine.

[0061] R¹ in the formnula R¹—X—Z is preferably an organic group. In apreferred embodiment, R¹ is an aromatic group, a heterocyclic group oran alkyl group. In a particularly preferred embodiment, R¹ is a C5-C10,a C5-C10 heterocyclic group and more preferably a C5-C9 heterocyclicgroup, or a C1-C18 alkyl group and more preferably a C1-C4 alkyl group.In one embodiment, R¹ is a fluoroalkyl.

[0062] As used herein, the term “organic group” refers to a hydrocarbongroup that is classified as an aliphatic group an aromatic group, acyclic group, or a combination of aliphatic and cyclic groups (e.g.,alkaryl and aralkyl groups). In the context of the present invention,the term “aliphatic group” refers to a saturated or unsaturated linearor branched hydrocarbon group. This term is used to encompass alkyl,alkenyl, and alkynyl groups, for example. The term “alkyl group” means asaturated linear or branched hydrocarbon group including, for example,methyl, ethyl, isopropyl, t-butyl, heptyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. The term “alkenyl group” refers to anunsaturated, linear or branched hydrocarbon group with one or morecarbon-carbon double bonds, such as a vinyl group. The term “alkynylgroup” refers to an unsaturated, linear or branched hydrocarbon groupwith one or more carbon-carbon triple bonds. The term “cyclic group”refers to a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” refers to mono- or polynuclear aromatic hydrocarbon group. Theterm “heterocyclic group” refers to a closed ring hydrocarbon in whichone or more of the atoms in the ring is an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.).

[0063] As is well understood in this technical area, a large degree ofsubstitution is not only tolerated, but is often advisable. Substitutionis anticipated on the compounds of the present invention. As a means ofsimplifying the discussion and recitation of certain terminology usedthroughout this application, the terms “group” and “moiety” are used todifferentiate between chemical species that allow for substitution orthat may be substituted and those that do not allow or may not be sosubstituted. Thus, when the term “group” is used to describe a chemicalsubstituent, the described chemical material includes the unsubstitutedgroup and that group with O, N, or S atoms, for example, in the chain aswell as carbonyl groups or other conventional substitution. Where theterm “moiety” is used to describe a chemical compound or substituent,only an unsubstituted chemical material is intended to be included. Forexample, the phrase “alkyl group” is intended to include not only pureopen chain saturated hydrocarbon alkyl substitutents, such as methyl,ethyl, propyl, t-butyl, and the like, but also alkyl substitutentsbearing further substituents known in the art, such as hydroxy, alkyoxy,alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxy., etc.. Thus,“alkyl group” includes ether groups, haloalkyls, fluoroalkyls,nitroalkyls, carboxyalkyls, hydroxyalkyls, sufloalkyls, etc. On theother hand, the phrase “alkyl moiety” is limited to the inclusion oronly pure open chain saturated hydrocarbon alkyl substitutents, such asmethyl, ethyl, propyl, t-butyl, and the like.

[0064] In the vasoactive peptides of this invention, having the generalformula R¹—X—Z, X is preferably

[0065] wherein R² and R³ are independently H or an organic group and nis a whole integer from about 1 to about 20, preferably a whole integerfrom about 5 to about 20 and more preferably a whole integer from about1 to about 4. In a preferred embodiment, R² and R³ are independently H,an aromatic group, or an alkyl group and in a particularly preferredembodiment, R² and R³ are independently H or a C5-C10 aromatic group ora C1-C18 alkyl group and more preferably a C1-C4 alkyl group. In yetanother preferred embodiment, R² and R³ are independently H, a loweralkyl moiety (e.g., about a C1-C4 alkyl) or a phenyl moiety.

[0066] In a preferred embodiment of this invention R¹ is an aromaticgroup, a heterocyclic group or an alkyl group and R² and R³ areindependently H, an aromatic group or an alkyl group. In one aspect ofthis embodiment, R¹ has the general formula:

[0067] In a preferred embodiment of this invention R⁴-R⁸ are eachindependently selected from the group of H, fluoro, chloro, bromo, iodo,nitro, nitrile (cyano), amino, N-methyl amino, N,N-dimethyl amino,hydroxy, methoxy, thiomethoxy (S-methyl), methyl, ethyl, n-propyl,iso-propyl, -butyl, iso-butyl, sec-butyl, tert-butyl, trifluoromethyl,trifluoromethoxy, vinyl, acetamido, benzyl, toluyl, and methoxybenzyl.In one preferred embodiment, R⁶ is trifluoromethyl and one or more ofR⁴, R⁵, R⁷ and R⁸ are F.

[0068] In a further embodiment, R¹ is monocyclic, including, forexample, both five-membered rings and six-member rings. A preferred fivemember ring with the general formula:

[0069] preferably includes Y as O, NH or S. The diagonal line extendingfrom the center of five-membered ring and from the center of the ringsof the structures depicted below indicates that the substituent —X—Z canbe covalently attached to the ring at any of the carbon atoms that formthe ring. In an embodiment where R¹ is a six-membered ring, preferablyR¹ has the formula:

[0070] and preferably X is CH or N.

[0071] In another embodiment of this invention the invention R¹ is abicyclic ring having one of the following formulas:

[0072] wherein X is either N or C.

[0073] Preferred peptide modifications of this invention include, butare not limited to N-α-benzyl-Z, and N-α-benzoyl-Z, wherein Z is apeptide fragment capable of binding to a CGRP receptor. Preferred benzylanalogues include, but are not limited to, N-2-furanyl-Z, N-3-furanyl-Z,N-2-pyrrolyl-Z, N-3-pyrrolyl-Z, N-2-thiophenyl-Z, N-3-thiophenyl-Z,N-2-pyridyl-Z, N-3-pyridyl-Z, N-4-pyridyl-Z, N-1-naphthyl-Z,N-2-naphthyl-Z, N-2-quinolinyl-Z, N-4-quinolinyl-Z, N-8-quinolinyl-Z,N-1-isoquinolinyl-Z, N-3-isoquinolinyl-Z, R-N-α-methylbenzyl-Z,S-N-α-methylbenzyl-Z, α,α-dimethylbenzyl-Z, N-diphenylmethyl-Z,N-trityl-Z, and [D-Phe⁰]-Z. Other preferred benzoyl analogues of thepeptide fragments of this invention include, but are not limited toN-2-furanoyl-Z, N-3-furanoyl-Z, N-2-pyrroloyl-Z, N-3-pyrroloyl-Z,N-2-thiophenoyl-Z, N-3-thiophenoyl-Z, N-2-thiophenoyl-Z,N-3-thiophenoyl-Z, N-2-pyridoyl-Z, N-3-pyridoyl-Z, N-4-pyridoyl-Z,N-1-naphthoyl-Z, N-2-naphthoyl-Z, N-2-quinolinoyl-Z, N-3-quinolinoyl-Z,N-4-quinolinoyl-Z, N-8-quinolinoyl-Z, N-i -isoquinolinoyl-Z, and N-3-isoquinolinoyl-Z. Still other preferred peptide modifications of thisinvention include N-methanesuphonyl-Z, N-trifluoromethanesulphonyl-Z,N-benzenesulphonyl-Z, N-toluenesulphonyl-Z,N-4-methoxybenzenesulphonyl-Z, N-mesitylenesulphonyl-Z,N-4-trifluorobenzenesulphonyl-Z andN-4-trifluoromethoxybenzenesulphonyl-Z.

[0074] Preferred vasoactive peptides fragments are peptide fragments ofCGRP with CGRP antagonist activity, that is the peptide fragments ofthis invention inhibit CGRP activity. Preferably the peptides inhibitCGRP activity by at least 25% and preferably inhibit CGRP activity by atleast 50%. Particularly preferred peptide fragments of this inventionare peptide fragments of α-CGRP or β-CGRP, and preferably human α-CGRPor β-CGRP. Preferred peptide fragments of CGRP include CGRP (8-37).

[0075] The peptides of this invention can be prepared using methodsknown in the art. Exemplary methods for preparing the peptides of thisinvention are provided in Example 1. For example, a number of peptidesaccording to this invention can be assembled on MBHA resin using themethodology of Smith, D. D. et al. J. Med. Chem. 36:2536-2541, 1993.Those of ordinary skill in the art of peptide and protein modificationcan prepare the other peptide modifications of this invention withoutundue experimentation.

[0076] In one embodiment of this invention, the invention relates to amethod for inhibiting CGRP binding to one or more CGRP receptors bycontacting an effective amount of a peptide of this invention with theCGRP receptor. This method can be used in vitro, for example in assaysto identify and/or isolate CGRP receptors or with intact cells either invitro or in vivo to inhibit the effects of CGRP binding to its receptor.As an example of an assay to determine the ability of the peptides ofthis invention to compete with CGRP receptors, Example 2 illustrates anassay to determine whether a particular peptide modification of thisinvention can inhibit CGRP binding to a CGRP receptor.

[0077] Binding assays used to identify whether or not a particularpeptide would inhibit CGRP binding to its receptor have been carried outusing rat brain (Dennis, et al., J. Pharmacol. Exp. Ther. 254:123-128,1990, van Rossum, et al. J. Pharmacol. Exp. Ther. 269:846-853, 1994),spleen (Dennis, et al. J. Pharmacol. Exp. Ther. 254:123-128, 1990) andvas deferens (Mimeault, et al. J. Pharmacol. Exp. Ther. 258:1084-1090,1991), guinea-pig atrium and vas derferens brain (Dennis, et al. J.Pharmacol. Exp. Ther. 254:123-128, 1990, van Rossum, et al. J.Pharmacol. Exp. Ther. 269:846-853, 1994), human neuroblastomer cellsSK-N-MC (Rist et al. J. Med. Chem. 41:117-123, 1998) and pig brain, lung(Aiyar et al. J. Neurochem. 65:1131-1138, 1995) and kidney (Aiyar et al.Endocrinology 129:965-969, 1991).

[0078] Alternatively, the modified peptides of this invention, includingthose disclosed in the examples, can be used to identify other CGRPreceptors or alternatively, the assays of the present invention can beused to test and compare the efficacy of other CGRP antagonists. Forexample, the K₁ for a specific peptide in binding to a specific type ofCGRP receptor is a constant. If the K₁ value for the same peptide isdifferent in one tissue compared to another tissue then this is evidencefor two different receptors in these tissues.

[0079] Where the peptides of this invention are used to identify otherCGRP antagonists, the peptides of this invention can be used incompetition assays with candidate antagonists for example, using eitherlabeled peptide or labeled candidate antagonist, to assess preferentialbinding of the receptor to a peptide of this invention or to a testantagonist. The peptides of this invention or labeled candidateantagonist can be radiolabeled, labeled with a fluorescent tag,biotinylated or otherwise tagged and/or labeled using methods known inthe art.

[0080] CGRP has been implicated in a variety of diseases and pathologiesas has been described in the background section of this disclosure. CGRPacts as an antagonist of insulin action and CGRP is a potentvasodilator. Activity of CGRP is mediated through binding of CGRP to oneor more CGRP receptors. For purposes of this disclosure, the termCGRP-receptor superfamily refers to the class of cell receptors thatbind CGRP. The CGRP antagonist, CGRP(8-37) is a known antagonist butdoes not appear to consistently bind strongly to one or more CGRPreceptors. Exemplary peptides of this invention have demonstrated anincrease in binding affinity of about 65 fold over that reported forCGRP(8-37).

[0081] CGRP receptor antagonists have been tested in vivo. For example,as indicated, CGRP(8-37) has been shown to reverse hypotension andtachycardia produced by bacterial lipopolysaccharide (LPS)administration. Therefore, this invention also relates to atherapeutically effective amount of the peptides of the presentinvention, preferably in a pharmaceutically acceptable buffer such asphosphate buffered solutions including saline as well as other bufferedsolutions well known in the art of pharmaceutical formulations that canbe administered to an animal, including humans, to limit or otherwiseinhibit the effects of CGRP binding to one or more CGRP receptors. Thepeptides can be delivered to the animal using a method that is suitablefor the pathology being treated including, but not limited to,intravascular routes of delivery, parenteral routes, where applicable,intramuscular routes, or through the airways using an aerosol, a drip,or the like.

[0082] All references and publications cited herein are expresslyincorporated by reference into this disclosure. Particular embodimentsof this invention will be discussed in detail and reference has beenmade to possible variations within the scope of this invention. Thereare a variety of alternative techniques and procedures available tothose of skill in the art which would similarly permit one tosuccessfully perform the intended invention.

EXAMPLE 1 Synthesis of CGRP(8-37) Analogues Chemicals and Materials

[0083] N-α-Butyloxycarbonyl (Boc) amino acid derivatives were purchasedfrom Bachem (Torrence, Calif.) and Applied Biosystems (Foster City,Calif.). Reactive side chains of amino acids were protected as follows:Arg, mesitylene-2-sulphonyl; Asp, benzyl ester; Cys, p-methoxybenzyl;His, benzyloxymethyl; Lys, 2-chlorocarbobenzoxy; Ser, benzyl ether; Thr,benzyl ether; Trp, formyl; Tyr, 2-bromocarbobenzoxy. Different batchesof para-methylbenzhydrylamine (p-MBHA) resin, from Applied Biosystems,were used with substitutions varying from 0.62-0.77 mmol/g. All solventsand reagents for peptide syntheses were peptide synthesis grade fromApplied Biosystems and Fisher Biotechnology (Pittsburgh, Pa.).Thioanisole, ethanedithiol (EDT), m-cresol and dimethylsulfide (DMS)were purchased from Aldrich (Milwaukee, Wis.), trifluoroacetic acid(TFA) and diethyl ether were from Fisher, trifluoromethanesulfonic acid(TFMSA) was from Applied Biosystems. All chemicals were used assupplied. Analytical and semi-preparative reversed-phase highperformance liquid chromatography (RP-HPLC) was performed on a WatersCorporation Inc. 625LC instrument. A VYDAC 218TP510 C₁₈ column (1×25 cm)supplied by the Nest Group (Southboro, Mass.) was used forsemi-preparative RP-HPLC. The flow rate was 4 ml/min and the eluant wascontinuously monitored at 230 nm and collected in 4 ml fractions.Analytical RP-HPLC was performed on a VYDAC 218TP5415 C₁₈, 300 Å, column(0.46×15 cm) and a KROMASIL C₈, 100 Å column (0.46×25 cm). The flow ratewas 1 ml/min and the eluant was continuously monitored at 220 nm. Waterwas obtained from a Barnstead Nanopure system and solvents for HPLC wereOptima grade from Fisher. TFA for HPLC was supplied by Pierce (Rockford,Ill.). Solvents used for RP-HPLC were, Solvent A: triethylammoniumphosphate (100 mM) pH 2.5; Solvent B: is a mixture of acetonitrile inSolvent A (60/40, vol/vol); Solvent C: 0.1% TFA in water; and Solvent D:0.09% TFA in acetonitrile/water (60/40, vol/vol). Electrosprayionization mass spectrometry (ESI-MS) was performed on all samples atthe Nebraska Center for Mass Spectrometry (Lincoln, Nebr.).

2. Solid Phase Peptide Synthesis of all Analogues.

[0084] All peptides were made by Merrifield's solid-phase methodology asdescribed previously (Smith, D. D. et a; J. Med. Chem.36:2536-2541,1993). N-α-Boc amino acid derivatives were coupled to p-MBHA resin (0.5meq.) in a four-fold excess using di-isopropylcarbodiimide andhydroxybenzotriazole in N-methylpyrrolidinone. The coupling reactionswere monitored by the quantitative ninhydrin test (Sarin, V. K. et al.,Anal. Biochem. 117: 147-157, 1981).

[0085] The Boc protecting group was removed using 33% TFA indichloromethane (DCM) for one minute and 50% TFA in DCM for 20 minutes.The first 21 amino acid derivatives were single-coupled and the last 15amino acid derivatives were double-coupled to maintain yields in excessof 99%. After the twentieth coupling, the peptide-resin was dried andhalf was used for the rest of the synthesis. Once the desired sequenceswere assembled the N-terminal Boc group was removed as described aboveand portions of the peptide resin were either benzylated using benzylbromide (10 eq.) and DIEA (10 eq.) in DCM for six hours or benzoylatedusing benzoic anhydride (10 eq.) and DIEA (10 eq.) in DCM for one hour.Peptides were freed of their side chain protecting groups and cleavedfrom the resin by the low-high TFMSA method of Tam (Tam, J. P. et al.,J. Am. Chem. Soc. 108: 5242-5251, 1986) to yield an ether precipitated,crude product which was purified immediately.

[0086] For benzoyl derivatives and analogues, the N-terminal group canbe acylated following previously described procedures (Stewart and Youngin Solid Phase Peptide Synthesis, 2^(nd) edition p. 73, Pierce ChemicalCo. Rockford, Ill.) using either the appropriate benzoic anhydride (10eq.), benzoyl chloride (10 eq.) or sulphonyl chloride (10 eq.) in thepresence of DIEA (10 eq.) or the appropriate benzoic acid (4 eq.) andcoupling reagents such as diisopropyl carbodiimide (4 eq.) andhydroxybenzotriazole (4 eq.). The acylation reaction can be monitoredusing the known quantitative ninhydrin test. The benzoylated peptidescan be cleaved from the resin and purified.

[0087] In one example, 2-chlorobenzyol-h-α-CGRP(8-37) is synthesizedusing 0.25 mmol peptide resin treated with TFA/DCM (1/1 vol/vol), washedDCM (5×1 min), mixed with DIEA/DCM (5/95 vol/vol) and washed with DCM(5×1 min). A solution of 2-chlorobenzoyl chloride (438 mg, 2.5 mmol) andDIEA (0.43 ml, 2.5 mmol) in DCM (20 ml) is mixed with the deprotectedpeptide-resin for 1 hour or until the acylation is complete by thequantitative ninhydrin test. The resin is then washed with DCM (5×1 min)and dried under reduced pressure. The peptide is cleaved from the resinand purified.

[0088] In another example, benzyl derivatives and analogues can beprepared by alkylating the N-terminal amino group using the appropriatebenzyl halide (generally bromo or chloro compounds) (10 eq.) in thepresence of DIEA (10 eq.). These reaction conditions produce a mixtureof the mono-benzylated and di-benzylated peptides, that can then becleaved from the resin and purified.

Purification of Exemplary Analogues

[0089] Analogue 1a: N-α-benzyl-h-α-CGRP (8-37) and analoguedibenzyl-h-α-CGRP(8-37). The crude product derived from benzylating 280mg of peptide resin, was dissolved in solvent A (supra) and loaded on tothe semi-preparative RP-HPLC column previously equilibrated with amixture of solvent A and solvent B (76/24 vol/vol). The column waseluted using a linear gradient increasing solvent B composition to 54%over 75 minutes to yield two major products. Fractions containing thefirst product to elute from the column were pooled, diluted with anequal volume of water and loaded onto the same semi-preparative RP-HPLCcolumn previously equilibrated with a mixture of solvent C and solvent D(67/33, vol/vol). The product was eluted from the column using a lineargradient increasing solvent D composition to 53% over 50 minutes.Fractions containing the product, as determined by analytical RP-HPLC,were pooled and lyophilized to yield 5.2 mg of a white fluffy powder.The product was >98% pure by analytical RP-HPLC. k′ values underisocratic conditions and measured masses, determined by ESI-MS, arelisted in Table 1.

[0090] Fractions containing the later eluting product were pooled,diluted with an equal volume of water and loaded onto thesemi-preparative RP-HPLC column previously equilibrated with a mixtureof solvent C and solvent D (64/36, vol/vol). This product was elutedfrom the column using a linear gradient increasing solvent D compositionto 56% over 50 minutes. Fractions containing only the desired product,as determined by analytical RP-HPLC, were pooled and lyophilized toyield 8.5 mg of a fluffy, white powder. The product was >98% pure byanalytical RP-HPLC and identified as dibenzyl-h-α-CGRP(8-37) by ESI-MS.k′ values under isocratic conditions and measured masses, determined byESI-MS, are provided in Table 1.

[0091] Analogue 1b: N-α-benzoyl-h-α-CGRP (8-37). The title compoundresulting from benzoylation of 200 mg of peptide resin, was purifiedfollowing the same methods described above for N-α-benzyl-h-α-CGRP(8-37). The product was eluted from the semi-preparative RP-HPLC columnusing a linear gradient of 31% to 51% solvent B over 50 minutes followedby a linear gradient of 35% to 55% solvent D over 50 minutes, to yield20 mg of the desired lyophilized product (19%). k′ values underisocratic conditions and measured masses, determined by ESI-MS, arelisted in Table 1 (below).

[0092] Analogue 2a: N-α-benzyl-h-β-CGRP (8-37) anddibenzyl-h-β-CGRP(8-37). N-α-benzyl-h-β-CGRP (8-37) anddibenzyl-h-β-CGRP(8-37) were obtained from benzylating 200 mg of peptideresin and purified following the same methods described above forN-α-benzyl-h-α-CGRP (8-37). Both products were eluted from thesemi-preparative RP-HPLC column using a linear gradient of 27% to 47%solvent B over 50 minutes. The first product to elute from the columnwas further purified on the semi-preparative column using a lineargradient of 31% to 51% solvent D over 50 minutes to yield 4 mg of awhite fluffy powder. The product was >98% pure by analytical RP-HPLC andidentified as N-α-benzyl-h-β-CGRP(8-37) by ESI-MS. k′ values underisocratic conditions and measured masses, determined by ESI-MS, arelisted in Table 1.

[0093] The later eluting product was further purified bysemi-preparative RP-HPLC using a linear gradient of 34% to 54% solvent Dover 50 minutes to yield 5.3 mg of a white, fluffy powder. The productwas >98% pure by analytical RP-HPLC and identified asdibenzyl-h-β-CGRP(8-37) by ESI-MS. k′ values under isocratic conditionsand measured masses, determined by ESI-MS are listed in Table 1.

[0094] Analogue 2b: N-α-benzoyl-h-β-CGRP (8-37). The title compound,derived from benzoylating 200 mg of peptide resin, was purifiedfollowing the same methods described above for N-α-benzyl-h-α-CGRP(8-37). The product was eluted from the semi-preparative RP-HPLC columnusing a linear gradient of 32% to 52% solvent B over 50 minutes followedby a linear gradient of 36% to 56% solvent D over 50 minutes to yield 10mg of the desired lyophilized product (10%). k′ values under isocraticconditions and measured masses, determined by ESI-MS, are listed inTable 1. TABLE 1 Physicochemical Properties of Analogues AnalyticalESI-MS RP-HPLC (k′) Calcu- Ob- ^(a)System Peptide lated served 1^(b)System 2 N-α-benzyl-h-α-CGRP (8-37) 3215.6 3215.6 0.6 5.6^(c)N-α-benzoyl-h-α-CGRP (8-37) 3229.6 3229.4 3.3 7.7 N-α-benzy1-h-β-CGRP(8-37) 3221.0 3221.3 0.7 2.0 N-α-benzoyl-h-β-CGRP (8-37) 3233.7 3233.54.3 4.1^(d) Dibenzyl-h-α-CGRP (8-37) 3305.5 3305.6 1.8 3.8Dibenzyl-h-β-CGRP (8-37) 3311.1 3310.7 1.8 4.7

EXAMPLE 2 CGRP Antagonist Testing 1. Membrane Preparations

[0095] Cell membranes were prepared from left circumflex, left anteriordescending and right circumflex epicardial coronary arteries dissectedfrom fresh pig hearts obtained from a local slaughterhouse. The arterieswere cleaned of surrounding fat and connective tissue. They were cutopen exposing the luminal surface and the endothelium was removed byrubbing. Arteries were then cross-cut into thin strips with a razorblade and homogenized in ice-cold 50 mM Tris-HCl buffer, pH 7.4,containing 5 mM EDTA (Na₂—Ca salt) using a Polytron (speed setting 5 for20 sec). The homogenate was centrifuged at 1600 g for 10 min at 4° C.The supernatant was collected, homogenized and crude membranes pelletedby high-speed centrifugation at 50,000 g for 30 min at 4° C. Themembrane pellet was reconstituted in ice cold 50 mM Tris-HCl, pH 7.4,containing 100 mM Nacl and 5 mM MgCl₂ and the steps of homogenizationand centrifugation repeated twice as described above. The dried membranepellet was then stored at −80° C. until use. Protein was determinedaccording to the method of Lowry (Lowry, O. H. et al., J. Biol. Chem.193:265-275, 1951)

Radioligand Binding Studies.

[0096] Crude membranes (50 μg membrane protein/tube) were incubated withvarying concentrations of drugs (h-α-CGRP (8-37), N-benzyl-h-α-CGRP(8-37) or N-benzoyl h-α-CGRP (8-37)) together with 50pM¹²⁵I-[His¹⁰]-h-α-CGRP for 50 min at 37° C. Incubations were performedin 50 mM Tris-HCl buffer, pH 7.4 containing 5 mM MgCl₂, 100 mM NaCl,0.1% (w/v) bovine serum albumin and 0.05% (w/v) bacitracin. Non-specificbinding was defined as binding remaining in the presence of 1 μMh-α-CGRP. Bound ¹²⁵I-h-α-CGRP was separated from free by vacuumfiltration (Brandel cell harvester, model MG-48R) through glass fiberfilters (Schliecher & Schuell, #32) and counted using a γ counter (LKBWallac 1277). To reduce non-specific binding of peptides to chargedsurfaces, glass incubation tubes were coated with Sigmacote and glassfiber filters soaked for 60 min in 0.2% (v/v) polyethyleneimine prior touse.

[0097] We established several criteria in order for each experiment tobe considered as valid data. These criteria stated that specific bindingof the radiolabel was greater than or equal to 70% and that theproportion of radiolabel bound to the membrane was less than 10% of thetotal amount added to the incubation.

Relaxation of Pig Coronary Arteries.

[0098] The proximal portion of the left circumflex coronary artery wasdissected from pig hearts at a local slaughterhouse and transported inice-cold Krebs' solution (composition in mM; NaCl 125, KCl5.5, CaCl₂2H₂O 2.5, MgCl₂ 6H₂0 1.2, NaH₂PO₄ 1.25, NaHCO₃ 25, dextrose 11. 1,Na₂Ca-EDTA 2H₂O 0.029), equilibrated with 95% O₂/5% CO₂. Arteries werecleaned of adhering fat and connective tissue. Rings (2 mm long) werecut and mounted between two stainless-steel pins passed through thelumen of the vessel, then placed in water-jacketed organ bathsmaintained at 37° C. Rings were bathed in Krebs solution gassed with 95%O₂/5% CO₂, pH 7.4. One pin was connected to a Grass FT.03 forcetransducer for measurement of isometric tension with a Grass model 7Dpolygraph (Quincy, Mass.). Coronary artery rings were equilibrated at 6g of resting tension (determined to be optimal in previouslength-tension experiments by Bockman, C. S. et al., J. Pharmacol. Exp.Ther. 267: 1126-1133, 1993) for 30 min and then challenged twice with 45mM KCl. To measure relaxation, tone was induced in the rings using asubmaximal dose of KCl (ca. 15 mM) and when the response reached astable degree of contractile tone, complete cumulativeconcentration-response relaxation curves for agonists were generated.EC₅₀ values (i.e., the concentration of analog needed to cause one-halfof maximal relaxation) were used to quantify the potency of agonists incausing relaxation and were calculated by non-linear regression of alldata points on the relaxation concentration-response curve.

Functional Determination of Antagonist Affinity Values.

[0099] To determine antagonist affinity values, coronary artery ringswere prepared, equilibrated and contracted as described above. In someexperiments, endogenous CGRP was depleted by incubating rings in Krebs'solution containing 100 μM capsaicin and 10 μM indomethacin for threehours. Indomethacin was added to prevent capsaicin-induced contractionof coronary arteries mediated through release of prostaglandins from theadventitia (Franc-Cereceda, A., et al. Eur. J. Pharmacol. 142: 235-243,1987). Rings were then washed extensively for one hour to removecapsaicin and indomethacin. Cumulative concentration-response curves forh-α-CGRP-induced relaxation were generated in all rings, and the ringswere then washed and re-equilibrated with Krebs' solution for 60 min.Control rings were incubated with Krebs solution only for 90 minfollowed by relaxation concentration-response curves for h-α-CGRP. Nochange in the potency of h-α-CGRP in causing relaxation was observedafter the 90 min incubation period in control arteries. Some rings werethen incubated with the antagonist, h-α-CGRP (8-37) for 90 min prior tobeginning concentration-response curves for h-α-CGRP-induced relaxation.Three adjacent rings from each animal were treated with differentconcentrations of antagonist. For each concentration of antagonist used,dose-ratios were calculated by dividing the EC₅₀ value forh-α-CGRP-induced relaxation in the presence of antagonist by its EC₅₀value in the absence of antagonist. Schild plots were constructed andlinear regression used to determine the X-intercept (pA₂ value). Theslopes of the Schild plots are expressed as the mean ±95% confidencelimit. Differences in the slopes of Schild plots were determined byanalysis of covariance. The individual pA₂ values were averaged andexpressed as mean K_(B) values by conversion to their antilogs.

[0100] The inhibition of binding of the peptides of Table 1, such as¹²⁵I-[His¹⁰]-h-α-CGRP (1-37) binding, to membranes prepared from pigcoronary arteries is shown in FIGS. 1A and 1B. Membranes were preparedfrom the left circumflex, left anterior descending and right circumflexcoronary arteries. Competition binding experiments were performed byincubating coronary artery membranes (50 μg protein/tube) with 50 pM¹²⁵I-[His¹⁰]-h-αCGRP and 16 different concentrations of cold ligand.Nonspecific binding was defined experimentally as the boundradioactivity remaining in the presence of 1 μM h-αCGRP or 1 μM h-βCGRP.Inhibition is expressed in the figures as percent of¹²⁵I-[His¹⁰]-h-αCGRP binding. The potency of each of these analogs incompeting for binding to CGRP receptors was determined from nonlinearregression analysis of all data points on the curve. The rank order ofpotency for the modified αCGRP peptides in inhibiting¹²⁵I-[His¹⁰]-h-αCGRP from these binding sites wasdibenzyl-h-αCGRP(8-37)>N-benzoyl-h-αCGRP(8-37)>N-benzyl-h-αCGRP(8-37)>H-αCGRP(8-37).The rank order of potency for the modified βCGRP peptides in inhibiting¹²⁵I-[His¹⁰]-h-αCGRP from these binding sites wasN-benzoyl-h-βCGRP(8-37)≧dibenzyl-h-βCGRP(8-37)>N-benzyl-h-βCGRP(8-37)>h-βCGRP(8-37).Each competition curve shown was the mean of 3 or 4 experiments, eachusing membranes prepared from different animals.

[0101] The IC₅₀ values for each of h-α-CGRP (8-37) modified peptides andh-β-CGRP (8-37) modified peptides are listed in Table 2 and Table 3,respectively. Since the results are similar for either the α- or β-form,only the α- will be discussed. The modified peptides possessed higheraffinity than the CGRP1 receptor selective antagonist, h-α-CGRP (8-37)or h-β-CGRP (8-37). Except for N-α-benzyl-h-α-CGRP (8-37), all of thesepeptides were able to compete with the radioligand for binding at asingle affinity site. In the case of N-α-benzyl-h-α-CGRP (8-37), Thiscompound appeared to compete for binding to high- and low-affinitybinding sites, suggesting the presence of two receptors.

[0102] Data were analyzed using non-linear least squares curve fitting(Graphpad Inplot). The concentration of unlabeled peptide required toinhibit the binding of 125I-[His¹⁰]-CGRP from half of these sites (IC₅₀)was taken as the measure of affinity of each of these peptides.Non-specific binding determined using 1 μM h-α-CGRP was not differentfrom non-specific binding determined from treating the minimum value ofthe competition curve as a fitted parameter. Therefore, in our analysisnon-specific binding was defined by the minimum value of the competitioncurve. Comparisons of one- and two-site fits of binding data were madeusing an F-test option. In cases where P<0.05, the two-site bindingmodel was accepted as the best fit of the data.

[0103] Results of the relaxation studies are illustrated in FIG. 2. Asprovided above, ring segments of pig left circumflex coronary arterywere cleaned and mounted in glass chambered organ baths filled withDrebs-Henseleit buffer, pH 7.4 maintained at 37° C. Isometric tension onthese ring segments was measured by force transducers and recorded on apolygraph. Cumulative concentration-response curves for h-αCGRP weregenerated on each segment in the presence and absence of antagonist. Themean concentration response curves from 4 experiments illustrating therightwards shifts of h-αCGRP-induced relaxation caused by increasingconcentrations of h-αCGRP(8-37) (FIG. 2A), N-benzoyl-h-αCGRP(8-37) (FIG.2B), N-benzyl-αCGRP(8-37) (FIG. 2C) and dibenzyl-h-αCGRP(8-37) (FIG. 2D)are shown. The shifts of the agonist concentration-response curvesproduced by each concentration of the antagonist were used to calculatedose-ratio values. Linear regression analysis of these dose-ratio valuesplotted on a log (DR-1) vs. log [antagonist] plot was used to determinethe affinity of the antagonist, denoted by the x-intercept of theregression.

[0104] N-benzyl-h-α-CGRP (8-37) and N-benzoyl-h-α-CGRP (8-37) bothinhibited h-α-CGRP-induced relaxation of isolated pig coronary arteryrings. Similar to h-α-CGRP (8-37), increasing concentrations of thesetwo compounds produced increasing rightwards shifts of the h-α-CGRPconcentration-response curve. Both compounds had higher affinity for theCGRP receptor in this tissue. Relative to the K_(B) of 970 nM forh-α-CGRP (8-37), the K_(B) for N-benzyl-h-α-CGRP (8-37) was 407 nM andthe K_(B) for N-benzoyl-h-α-CGRP (8-37) was 55 nM. Due to their higheraffinity in blocking h-α-CGRP responses than CGRP(8-37), both theseantagonists can replace CGRP(8-37). TABLE 2 Radioligand Binding Data forh-α-CGRP (8-37) modified analogs Analogue Affinity IC₅₀ (nM) p h-α-CGRP(8-37) 14.38 ± 0.38  N-benzyl-h-α-CGRP (8-37) 1.58 ± 0.38 0.004N-benzoyl-h-α-CGRP (8-37) 0.27 ± 0.0  <0.0001 dibenzyl-h-αCGRP(8-37)0.22 ± 0.06 <0.0001

[0105] TABLE 2A Inhibition of Relaxation for h-α-CGRP(8-37) modifiedanalogs Peptide Antagonist K_(B) nM p h-α-CGRP (8-37) 970.1 ± 300N-benzyl-h-α-CGRP (8-37) 118.7 ± 56.4 <0.0001 N-benzoyl-h-α-CGRP (8-37)40.36 ± 18.95 <0.0001 dibenzyl-h-αCGRP(8-37) 29.02 ± 6.37 <0.0001

[0106] TABLE 3 Radioligand Binding Data for h-β-CGRP (8-37) modifiedanalogs Analogue Affinity IC₅₀ (nM) p h-β-CGRP (8-37) 20.65 ± 3.96N-benzyl-h-β-CGRP (8-37)  3.62 ± 0.8 0.0004 N-benzoyl-h-β-CGRP (8-37) 0.63 ± 0.09 <0.0001 dibenzyl-h-βCGRP(8-37)  0.73 ± 0.14 <0.0001

EXAMPLE 3 Antagonist Testing in Rats

[0107] In vivo testing of these peptides uses anesthetized rats. Acannula is placed in the right carotid artery for measurement of bloodpressure and a second cannula is placed in the left femoral vein and isused for injection of peptides into the circulation. CGRP is injectedfirst and a reduction in blood pressure has been reported by others(Fisher et al. Nature 305:534-536, 1983). After blood pressure returnsto normal a CGRP antagonist is given followed by a second injection ofCGRP. Inhibition of the hypotensive effect of CGRP by the antagonist isevidence that the reduction in blood pressure is mediated by CGRPreceptors.

[0108] While particular embodiments of the invention have been describedin detail, it will be apparent to those skilled in the art that theseembodiments are exemplary rather than limiting, and the true scope ofthe invention is that defined in the following claims.

1 23 29 amino acids amino acid single linear peptide 1 Thr His Arg LeuAla Gly Leu Leu Ser Arg Ser Gly Gly Met Val Ly 1 5 10 15 Ser Asn Phe ValPro Thr Asn Val Gly Ser Lys Ala Phe 20 25 30 amino acids amino acidsingle linear peptide 2 Thr His Arg Leu Ala Gly Leu Leu Ser Arg Ser GlyGly Met Val Ly 1 5 10 15 Ser Asn Phe Val Val Pro Thr Asn Val Gly Ser LysAla Phe 20 25 30 37 amino acids amino acid single linear peptide 3 AlaCys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15 SerArg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Va 20 25 30 GlySer Lys Ala Phe 35 37 amino acids amino acid single linear peptide 4 AlaCys Asp Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15 SerArg Ser Gly Gly Val Val Lys Asn Asn Phe Val Pro Thr Asn Va 20 25 30 GlySer Lys Ala Phe 35 37 amino acids amino acid single linear peptide 5 SerCys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15 SerArg Ser Gly Gly Val Val Lys Asp Asn Phe Val Pro Thr Asn Va 20 25 30 GlySer Lys Ala Phe 35 37 amino acids amino acid single linear peptide 6 SerCys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15 SerArg Ser Gly Gly Val Val Lys Asp Asn Phe Val Pro Thr Asn Va 20 25 30 GlySer Glu Ala Phe 35 37 amino acids amino acid single linear peptide 7 AlaCys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Asp Phe Le 1 5 10 15 SerArg Ser Gly Gly Val Gly Lys Asn Asn Phe Val Pro Thr Asn Va 20 25 30 GlySer Lys Ala Phe 35 37 amino acids amino acid single linear peptide 8 GlyCys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15 SerArg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Va 20 25 30 GlySer Glu Ala Phe 35 37 amino acids amino acid single linear peptide 9 SerCys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15 SerArg Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asp Va 20 25 30 GlySer Glu Ala Phe 35 37 amino acids amino acid single linear peptide 10Ser Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 10 15Ser Arg Ser Gly Gly Val Val Lys Ser Asn Phe Val Pro Thr Asn Va 20 25 30Gly Ser Gln Ala Phe 35 37 amino acids amino acid single linear peptide11 Ser Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Le 1 5 1015 Ser Arg Ser Gly Gly Val Val Lys Ser Asn Phe Val Pro Thr Asn Va 20 2530 Gly Ser Glu Ala Phe 35 37 amino acids amino acid single linearpeptide 12 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala Asp PheLe 1 5 10 15 Asn Arg Ser Gly Gly Met Gly Asn Ser Asn Phe Val Pro Thr AsnVa 20 25 30 Gly Ala Lys Ala Phe 35 37 amino acids amino acid singlelinear peptide 13 Ala Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu AlaAsp Phe Le 1 5 10 15 Ser Arg Ser Gly Gly Met Ala Lys Asn Asn Phe Val ProThr Asn Va 20 25 30 Gly Ser Lys Ala Phe 35 52 amino acids amino acidsingle linear peptide 14 Tyr Arg Gln Ser Met Asn Asn Phe Gln Gly Leu ArgSer Phe Gly Cy 1 5 10 15 Arg Phe Gly Thr Cys Thr Val Gln Lys Leu Ala HisGln Ile Tyr Gl 20 25 30 Phe Thr Asp Lys Asp Lys Asp Asn Val Ala Pro ArgSer Lys Ile Se 35 40 45 Pro Gln Gly Tyr 50 50 amino acids amino acidsingle linear peptide 15 Tyr Arg Gln Ser Met Asn Gln Gly Ser Arg Ser ThrGly Cys Arg Ph 1 5 10 15 Gly Thr Cys Thr Met Gln Lys Leu Ala His Gln IleTyr Gln Phe Th 20 25 30 Asp Lys Asp Lys Asp Gly Met Ala Pro Arg Asn LysIle Ser Pro Gl 35 40 45 Gly Tyr 50 37 amino acids amino acid singlelinear peptide 16 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu AlaAsn Phe Le 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser SerThr Asn Va 20 25 30 Gly Ser Asn Thr Tyr 35 37 amino acids amino acidsingle linear peptide 17 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg LeuAla Asn Phe Le 1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro Val Leu ProPro Thr Asn Va 20 25 30 Gly Ser Asn Thr Tyr 35 31 amino acids amino acidsingle linear peptide 18 His Ala Thr Gln Arg Leu Ala Asn Phe Leu Val HisSer Ser Asn As 1 5 10 15 Phe Gly Ala Ile Leu Ser Ser Thr Asn Val Gly SerAsn Thr Tyr 20 25 30 30 amino acids amino acid single linear peptide 19Ala Thr Gln Arg Leu Ala Asn Phe Leu Val Arg Ser Ser Asn Asn Le 1 5 10 15Gly Pro Val Leu Pro Pro Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30 25amino acids amino acid single linear peptide 20 Val Leu Gly Lys Leu SerGln Glu Leu His Lys Leu Gln Thr Tyr Pr 1 5 10 15 Arg Thr Asn Thr Gly SerAsn Thr Tyr 20 25 24 amino acids amino acid single linear peptide 21 LeuGly Arg Leu Ser Gln Glu Leu His Arg Leu Gln Thr Tyr Pro Ar 1 5 10 15 ThrAsn Thr Gly Ser Asn Thr Tyr 20 25 amino acids amino acid single linearpeptide 22 Ala Thr Gln Arg Leu Ala Asn Glu Leu Val Arg Leu Gln Thr TyrPr 1 5 10 15 Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25 31 amino acidsamino acid single linear peptide 23 Thr Val Gln Lys Leu Ala His Gln IleTyr Gln Phe Thr Asp Lys As 1 5 10 15 Lys Asp Asn Val Ala Pro Arg Ser LysIle Ser Pro Gln Gly Tyr 20 25 30

What is claimed is:
 1. A vasoactive peptide having the general formula:R¹—X—Z wherein Z is a vasoactive peptide, R1 is an organic group

and wherein R2 and R3 are independently H or an organic group and n is awhole integer between 1 and
 10. 2. The peptide of claim 1 wherein Z is apeptide fragment of at least 15 amino acids from CGRP.
 3. The peptide ofclaim 2 wherein Z comprises the amino acid sequence of SEQ ID NO:1 orSEQ ID NO:2.
 4. The peptide of claim 2 wherein Z is an antagonist ofhuman CGRP.
 5. The peptide of claim 2 wherein Z is an antagonist ofα-CGRP or β-CGRP.
 6. The peptide of claim 1 wherein Z is a CGRPantagonist peptide fragment selected from the group consisting ofamylin, CGRP and adrenomedullin.
 7. The peptide of claim 5 wherein Zcomprises the amino acid sequence of SEQ ID NOS:6-17 and
 23. 8. Thepeptide of claim 5 wherein Z comprises the amino acid sequence of SEQ IDNOS 18-22.
 9. The peptide of claim 1 wherein R¹ is an aromatic group, aheterocyclic group or an alkyl group and R² and R³ are independently H,an aromatic group or an alkyl group.
 10. The peptide of claim 9 whereinR¹ is a C1-C4 alkyl group.
 11. The peptide of claim 10 wherein R¹ is afluoroalkyl.
 12. The peptide of claim 10 wherein R² and R³ areindependently H, a C1-C4 alkyl group or a phenyl moiety.
 13. The peptideof claim 10 wherein R¹ is a C5-C10 aromatic group, a C5-C9 heterocyclicgroup or a C1-C4 alkyl group.
 14. The peptide of claim 13 wherein R₂ andR₃ are independently H or a C5-C10 aromatic group or a C1-C4 alkylgroup.
 15. The peptide of claim 9 wherein R¹ has the general formula:

and wherein R⁴-R⁸ are each independently selected from the group of H,fluoro, chloro, bromo, iodo, nitro, nitrile (cyano), amino, N-methylamino, N,N-dimethyl amino, hydroxy, methoxy, thiomethoxy (S-methyl),methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,tert-butyl, trifluoromethyl, trifluoromethoxy, vinyl, acetamido, phenyl,toluyl, and methoxyphenyl.
 16. The peptide of claim 15 wherein R⁶ istrifluoromethyl and R⁴, R⁵, R⁷ and R⁸ are F.
 17. The peptide of claim 15wherein R¹ is

and wherein Y is selected from the group consisting of O, NH, and S. 18.The peptide of claim 9 wherein R¹ is selected from the group consistingof:

and wherein X is selected from the group consisting of C and N.
 19. Thepeptide of claim 15 wherein the peptide is a CGRP antagonist and apeptide or polypeptide of at least consecutive 15 amino acids selectedfrom a protein from the group consisting of N-α-benzoyl-α-CGRP,N-α-benzyl-β-CGRP, N-α-benzoyl-β-CGRP and N-α-benzyl-α CGRP,dibenzyl-h-α-CGRP and dibenzyl-h-β-CGRP.
 20. A method for inhibitingCGRP binding to one or more CGRP receptors comprising the step ofcontacting an effective amount of a composition comprising a peptideaccording to claim 4 with a CGRP receptor.
 21. The method of claim 20wherein the CGRP receptor is on a cell.
 22. The method of claim 20wherein the CGRP receptor is cell free.
 23. The method of claim 21wherein the cell is in culture.
 24. The method of claim 21 wherein thecell is part of a tissue.
 25. The method of claim 21 wherein the cell isin an animal.
 26. The method of claim 25 wherein the animal is a human.27. An assay for identifying CGRP antagonists comprising the step of:combining a peptide according to claim 4 with at least one CORP receptorand a test CGRP antagonist with at least one CGRP receptor; andcomparing binding of the peptide to the CGRP receptor with binding ofthe test antagonist to the CGRP receptor, wherein improved binding ofthe test antagonist to the CGRP receptor in the presence of the peptideof claim 4 identifies a candidate CGRP antagonist.
 28. A method foridentifying a CGRP receptor in a cell sample comprising the steps of:contacting a peptide of claim 4 with a cell sample to detect binding ofthe peptide to the cell; and isolating one or more receptors binding thepeptide to the cell.
 29. A method for inhibiting CGRP binding to one ormore CGRP receptors comprising contacting a CGRP receptor with aneffective amount of a composition comprising a peptide having thegeneral formula: R¹—X—Z wherein Z is a vasoactive peptide, R¹ is anorganic group, X is

and wherein R² and R³ are independently H or an organic group and n is awhole integer between 1 and
 10. 30. The method of claim 29 wherein Z isa peptide fragment of at least 15 amino acids from CGRP.
 31. The methodof claim 30 wherein Z comprises the amino acid sequence of SEQ ID NO:1or SEQ ID NO:2.
 32. The method of claim 30 wherein Z is an antagonist ofhuman CGRP.
 33. The method of claim 30 wherein Z is an antagonist ofα-CGRP or β-CGRP.
 34. The method of claim 33 wherein Z comprises theamino acid sequence of SEQ ID NOS:6-17 and
 23. 35. The method of claim33 wherein Z comprises the amino acid sequence of SEQ ID NOS:18-22. 36.The method of claim 29 wherein Z is a CGRP antagonist peptide fragmentselected from the group consisting of amylin, CGRP and adrenomedullin.37. The method of claim 29 wherein R¹ is an aromatic group, aheterocyclic group or an alkyl group and R² and R³ are independently H,an aromatic group or an alkyl group.
 38. The method of claim 37 whereinR¹ is a C1-C4 alkyl group.
 39. The method of claim 38 wherein R¹ is afluoroalkyl.
 40. The method of claim 38 wherein R² and R³ areindependently H, a C1-C4 alkyl group or a phenyl moiety.
 41. The methodof claim 38 wherein R¹ is a C5-C10 aromatic group, a C5-C9 heterocyclicgroup or a C1-C4 alkyl group.
 42. The method of claim 41 wherein R² andR³ are independently H or a C5-C10 aromatic group or a C1-C4 alkylgroup.
 43. The method of claim 37 wherein R¹ has the general formula:

and wherein R⁴-R⁸ are each independently selected from the group of H,fluoro, chloro, bromo, iodo, nitro, nitrile (cyano), amino, N-methylamino, N,N-dimethyl amino, hydroxy, methoxy, thiomethoxy (S-methyl),methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,tert-butyl, trifluoromethyl, trifluoromethoxy, vinyl, acetamido, phenyl,toluyl, and methoxyphenyl.
 44. The method of claim 43 wherein R⁶ istrifluoromethyl and R⁴, R⁵, R⁷ and R⁸ are F.
 45. The method of claim 43wherein R¹ is

and wherein Y is selected from the group consisting of O, NH, and S. 46.The method of claim 43 wherein the peptide is a CGRP antagonist and apeptide or polypeptide of at least 15 consecutive amino acids selectedfrom a protein from the group consisting of N-α-benzoyl-α-CGRP,N-α-benzyl-β-CGRP, N-α-benzoyl-β-CGRP and N-α-benzyl-α CGRP,dibenzyl-h-α-CGRP and dibenzyl-h-β-CGRP.
 47. The method of claim 37wherein R¹ is selected from the group consisting of:

and wherein X is selected from the group consisting of C and N.
 48. Anassay for identifing CGRP antagonists comprising: combining a peptidehaving the general formula: R¹—X—Z wherein Z is a vasoactive peptide, R¹is an organic group, X is

and wherein R² and R³ are independently H or an organic group and n is awhole integer between 1 and 10, with at least one CGRP receptor and atest CGRP antagonist with at least one CGRP receptor; and comparingbinding of the peptide to the CGRP receptor with binding of the testantagonist to the CGRP receptor, wherein improved binding of the testantagonist to the CGRP receptor in the presence of the peptideidentifies a candidate CGRP antagonist.
 49. The assay of claim 48wherein Z is a peptide fragment of at least 15 amino acids from CGRP.50. The assay of claim 49 wherein Z is an agatonist of human CGRP.
 51. Amethod for identifying a CGRP receptor in a cell sample comprising:contacting a peptide having the general formula: R¹—X—Z wherein Z is avasoactive peptide, R¹ is an organic group, X is

and wherein R² and R³ are independently H or an organic group and n is awhole integer between 1 and 10, with a cell sample to detect binding ofthe peptide to the cell; and isolating one or more receptors binding thepeptide to the cell.
 52. The assay of claim 51 wherein Z is a peptidefragment of at least 15 amino acids from CGRP.
 53. The assay of claim 51wherein Z is an agatonist of human CGRP.